Potentiometric titration of cytochrome-bo type quinol oxidase of Escherichia coli: evidence for heme-heme and copper-heme interaction

The cytochrome-bo quinol oxidase of Escherichia coli contains a high-spin b-type heme (cytochrome o), a low-spin b-type heme (cytochrome b) and copper. The EPR signal from cytochrome o is axial high spin and when titrated potentiometrically gives a bell-shaped curve. The low-potential side of this curve (Em7 approx. 160 mV) corresponds to the reduction/oxidation of the cytochrome. The high-potential side (Em7 approx. 350 mV) is proposed to be due to reduction/oxidation of a copper center; in the CuII form tight cytochrome o-copper spin coupling results in a net even spin system and loss of the EPR spectrum. Optical spectra of the alpha-bands of the reduced cytochromes at 77 K show that cytochrome b has its maxima at 564 nm when cytochrome o is oxidized but that this shifts to 561 nm when cytochrome o (max. 555 nm) is reduced. Both a heme-copper (cytochrome o-CuII) and a heme-heme (cytochrome o-cytochrome b) interaction are indicated in this quinol oxidase. These results indicate that cytochrome-bo quinol oxidase has a binuclear heme-copper catalytic site and suggest striking structural similarity to subunit I of the cytochrome aa3 system.     

Shigella flexneri infection is dependent on villin in the mouse intestine and in primary cultures of intestinal epithelial cells

Villin is an actin-binding protein present in intestinal and kidney brush borders. Villin has been shown to present in vitro Ca(2+)-dependent bundling and severing F-actin properties. The study of villin knock-out mice allowed us to show that while bundling of F-actin microfilaments is unaffected, this protein is important for the reorganization of the actin cytoskeleton elicited by various signals during both physiological and pathological conditions. Here, we studied the role of villin during infection by Shigella flexneri, the causative agent of bacillary dysentery. This bacterium induces the reorganization of the host actin cytoskeleton to penetrate into epithelial cells and spread from cell to cell. In vivo, we show that unlike newborn vil+/+ mice, which are sensitive to Shigella invasion, resulting in a destructive inflammatory response of the intestinal mucosa following intragastric inoculation, newborn vil-/- mice appear fully resistant to infection. Using primary cultures of intestinal epithelial cells derived from vil+/+ or vil -/- mice, we demonstrate that villin plays an essential role in S. flexneri entry and cell-to-cell dissemination. Villin expression is thus critical for Shigella infection through its ability to remodel the actin cytoskeleton.     

N-terminal control of small heat shock protein oligomerization: changes in aggregate size and chaperone-like function

The small heat shock protein superfamily is composed of proteins from throughout the phylogenetic spectrum that are induced upon environmental stress. Their structural stability under stress derives in large part from the central region of the proteins, which forms two beta sheets held together by hydrophobic interactions and appears to be present in all superfamily members. The length, sequence, and amino acid composition of the N- and C-terminals, in contrast, are quite variable. The role of the N-terminal has been hypothesized to control species-specific assembly of subunits into higher level structures. To test this, a set of constructs was designed and expressed: the N-terminal sequences preceding the start of the core regions of alphaA-crystallin and HSP 16.5 from Methanococcus jannaschii were swapped; the N-terminal of each protein was removed, and replaced with a brief N-terminal extension sequence; and two nonsense N-terminal sequences of approximately the same length and hydropathicity as the original replaced the alphaA-crystallin N-terminal. All constructs, plus the original recombinant sequences, could be overexpressed except for the 16.5 N-terminal extension, and all showed chaperone-like activity except for the hybrid with the 16.5 C-terminal. Size and properties of the replacement N-terminal place limits on aggregate size. Additional restrictions are imposed by the structure of the dimer.     

NADPH analog binding to constitutive nitric oxide activates electron transfer and NO synthesis.

We report here that NADPH analogs such as 2'5'ADP, ATP, and 2'AMP paradoxically activate constitutive calcium/calmodulin regulated nitric oxide synthases (cNOS), including the endothelial isoform (eNOS) and the neuronal isoform (nNOS). These activators compete with NADPH by filling the binding site of the adenine moiety of NADPH, but do not occupy the entire NADPH binding domain. Effects of these analogs on cNOS's include increasing the electron transfer rate to external acceptors, as assessed by cytochrome c reductase activity in the absence of calmodulin. In addition, NO synthase activity in the presence of calmodulin (with or without added calcium) was increased by the addition of NADPH analogs. In contrast, the same NADPH analogs inhibit iNOS, the calcium insensitive inducible isoform, which lacks control elements found in constitutive isoforms. Because ATP and ADP are among the effective activators of cNOS isoforms, these effects may be physiologically relevant.     

Structural bistability of the GAL regulatory network and characterization of its domains of attraction

Bistability is a system-level property, exploited by many biomolecular interaction networks as a key mechanism to accomplish different cellular functions (e.g., differentiation, cell cycle, switch-like response to external stimuli). Bistability has also been experimentally found to occur in the regulatory network of the galactose metabolic pathway in the model organism Saccharomyces cerevisiae. In this yeast, bistability generates a persistent memory of the type of carbon source available in the extracellular medium: under the same experimental conditions, cells previously grown with different nutrients generate different responses and get stably locked into two distinct steady states. The molecular interactions of the GAL regulatory network have been thoroughly dissected through wet-lab experiments; thus, this system provides a formidable benchmark to our ability to characterize and reproduce in silico the behavior of bistable biological systems. To this aim, a number of models have been proposed in the literature; however, we found that they are not able to replicate the persistent memory behavior observed in (Acar et al., 2005 ). The present study proposes a novel model of the GAL regulatory network, which, in addition to reproducing in silico the experimental findings, can be formally analyzed for structural multistability of the network, using chemical reaction network theory (CRNT), and allows the characterization of the domains of attraction (DA). This work provides further insights into the GAL system and proposes an easily generalizable approach to the study of bistability-associated behaviors in biological systems.     

Insulin receptor isoforms are differently expressed during human osteoblastogenesis

The reciprocal influence and bidirectional cross-talk between bone and energy metabolism is a recent finding, since the discovery that the product of osteoblasts osteocalcin increases pancreatic β-cell proliferation, insulin secretion and sensitivity. Conversely, the anabolic effect of insulin is crucial for osteoblast function, as suggested by severe osteopenia and increased incidence of fracture in insulin-deficient diabetic patients. The Insulin Receptor (IR) tyrosine kinase, which is commonly expressed in the insulin-sensitive liver, muscle, and adipose tissues, is also found in animal and human bone. Here we show that in human bone two insulin receptor isoforms (IR-A and IR-B) are differently expressed. Mature human osteoblasts predominantly express IR-B, whereas IR-A is mainly expressed in osteoblast precursors, and IR-B/IR-A mRNA ratio significantly increases along the osteogenic differentiation of mesenchymal stromal precursors. Moreover, transfected osteoprogenitors overexpressing IR-A show an increased proliferation rate. In contrast, when transfected with and overexpressing IR-B, their proliferation rate is reduced, corresponding to a more differentiated phenotype. In conclusion, the fine regulation of the expression of different isoforms of IR during osteogenic differentiation confirms the important role played by IR in bone homeostasis, providing the basis for new perspectives on the various involvements of IR isoforms in bone pathophysiology.     

Evidence for an inositol hexakisphosphate-dependent role for Ku in mammalian nonhomologous end joining that is independent of its role in the DNA-dependent protein kinase

Nonhomologous end-joining (NHEJ) is an important pathway for the repair of DNA double-strand breaks (DSBs) and plays a critical role in maintaining genomic stability in mammalian cells. While Ku70/80 (Ku) functions in NHEJ as part of the DNA-dependent protein kinase (DNA-PK), genetic evidence indicates that the role of Ku in NHEJ goes beyond its participation in DNA-PK. Inositol hexakisphosphate (IP₆) was previously found to stimulate NHEJ in vitro and Ku was identified as an IP₆-binding factor. Through mutational analysis, we identified a bipartite IP₆-binding site in Ku and generated IP₆-binding mutants that ranged from 1.22% to 58.48% of wild-type binding. Significantly, these Ku IP₆-binding mutants were impaired for participation in NHEJ in vitro and we observed a positive correlation between IP₆ binding and NHEJ. Ku IP₆-binding mutants were separation-of-function mutants that bound DNA and activated DNA-PK as well as wild-type Ku. Our observations identify a hitherto undefined IP₆-binding site in Ku and show that this interaction is important for DSB repair by NHEJ in vitro. Moreover, these data indicate that in addition to binding of exposed DNA termini and activation of DNA-PK, the Ku heterodimer plays a role in mammalian NHEJ that is regulated by binding of IP₆.     

Estradiol increases IRS-1 gene expression and insulin signaling in breast cancer cells.

This study demonstrates how the potentiating effects of E2 on insulin signaling in ER-positive breast cancer cells are consequent to an enhanced IRS-1 expression [corrected]. It induces an increase of both PI-3K/AKT and ERK1/2 activities. A direct action of E2 in the regulating mouse IRS-1 gene is also investigated in both Chinese hamster ovary and MCF-7 cells that are transfected with mouse IRS-1 regulatory sequences. The authors have reported, for the first time, how E2 induction of IRS-1 mRNA was correlated with a direct positive regulatory role of E2 on the IRS-1 promoter. This effect seems to be not strictly related to the cell type.